DNA cleavage activities and mechanism of a novel heterodinuclear macrocyclic complex

Article abstract of DOI:10.1016/j.inoche.2009.12.013

A new unsymmetrical bis-furan pendant-armed macrocyclic heterodinuclear Cu(II)Zn(II) complex, [CuZnL(OAc)](ClO₄)·3MeCN·H₂O(1) (H₂L was derived from the condensation between 1,6-bis(2-furyl)-2,5-bis(2-hydroxy-3-formyl-5-bromobenzyl)-2, 5-diazahexane and 1,3-diaminopropane), has been synthesized and characterized by EDS (energy dispersive spectroscopy) and X-ray crystallography determination. The interaction between the complex and calf thymus (CT) DNA was investigated by UV-vis absorption, viscosity experiment, fluorescent and CD spectroscopy. The mechanism of the cleavage of supercoiled DNA (pBR 322DNA) process was also studied. It is proposed that DNA cleavage promoted by the complex occurrence via oxidative mechanism.

Full text of DOI:10.1016/j.inoche.2009.12.013

Inorganic Chemistry Communications 13 (2010) 314–318
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
DNA cleavage activities and mechanism of a novel heterodinuclear
macrocyclic complex
a
a
Yuan He a, Xiaohui Wang a, Hong Zhou a, Zhiquan Pan a,b, , Junbo Li , Qimao Huang
*
a Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of Technology, Wuhan 430073, PR China
b State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
new unsymmetrical bis-furan pendant-armed macrocyclic heterodinuclear Cu(II)Zn(II) complex,
Received 3 November 2009
Accepted 9 December 2009
Available online 16 December 2009
[CuZnL(OAc)](ClO4)ꢀ3MeCNꢀH2O(1) (H2L was derived from the condensation between 1,6-bis(2-furyl)-
2,5-bis(2-hydroxy-3-formyl-5-bromobenzyl)-2, 5-diazahexane and 1,3-diaminopropane), has been syn-
thesized and characterized by EDS (energy dispersive spectroscopy) and X-ray crystallography determi-
nation. The interaction between the complex and calf thymus (CT) DNA was investigated by UV–vis
absorption, viscosity experiment, fluorescent and CD spectroscopy. The mechanism of the cleavage of
supercoiled DNA (pBR 322DNA) process was also studied. It is proposed that DNA cleavage promoted
by the complex occurrence via oxidative mechanism.
Keywords:
Heterodinuclear complex
Binding-constants
Viscosity
Ó 2010 Elsevier B.V. All rights reserved.
DNA cleavage
The chemistry of multi-metal complexes as artificial nucleases
has received much attention because of their generally higher
DNA cleavage activity [1–3], and their importance in cancer ther-
apy [4–6] and molecular biology [7,8]. According to the literatures,
macrocyclic ligands with the rigid groups showed a better DNA
cleavage ability after combining to the metal ions, because it
may involve a synergistic mechanism among metal ions and rigid
macrocyclic ligands [9].
dry acetonitrile, Cu(II)Pb(II) complex was synthesized by reacting
intermediate (1) with lead(II) perchlorate trihydrate (1 equiv.) in
dry acetonitrile, followed by adding dropwise a solution of 1,3-
diaminopropane(1 equiv.) in ethanol. The Cu(II)Zn(II) complex
were obtained by the transmetalation reaction between the
Cu(II)Pb(II) complex and Zinc(II) sulfate in ethanol solution. The li-
gand L1 and the complex were fully characterized by elemental
analysis, IR, 1H NMR, and EDS, respectively [16].
Although the DNA cleavage activities and mechanisms of a few
homodinuclear macrocyclic complexes [10,11] and some heterodi-
nuclear uncyclic complexes [12–14] have been studied, the DNA
cleavage studies of the heterodinuclear complexes of tetraazamac-
rocyclic dicompartmental ligands have not been reported yet.
Herein, in order to find the more efficient DNA cleavage agent
and discuss the synergistic effect in the processes of the cleavage,
we have designed and synthesized a heterodinuclear Cu(II)Zn(II)
complex of tetraazamacrocyclic dicompartmental ligand with
two furan pendants. To our knowledge, this work provides the first
investigation on the DNA cleavage activities and mechanism of a
new heterodinuclear macrocyclic complex.
The precursor L1 was prepared through the Mannich reaction
[15] of 5-bromosalicyladehyde, N,N0-bis(2-furyl)-1,2-diaminoe-
thane and paraformaldehyde (Scheme 1). Acyclic intermediate
(1) was obtained as a green precipitate by the reaction between
precursor (L1) and copper(II) acetate monohydrate (1 equiv.) in
The crystal structure of the title complex ([CuZnL(OAc)]
(ClO4)ꢀ3MeCNꢀH2O) is illustrated in Fig. 1. The structure of the com-
plex shows that the Cu(II) resides at the N(imine)2O2 site and the
Zn(II) at the N(amine)2O2 site, respectively. The two metal ions are
bridged by one acetate group and two phenolic oxygen atoms, and
each metal ion exhibits a distorted square-pyramidal coordination
configuration. The equatorial Cu-donor bond distances are in a
range of 1.927(3)–1.972(5) Å, while the axial Cu–O3 bond is
2.408(4) Å due to Jahn–Teller effect. The basal Zn-donor bond dis-
tances are in a range of 2.033(3)–2.117(4) Å, and the apical Zn–O4
bond is 1.944(4) Å. The dihedral angle between the N(amine)2O2
and N(imine)2O2 plane is 16.4°. The deviation of Zn(II) and Cu(II)
from the relative basal planes are 0.6182 and 0.1049 Å, respectively.
Two furyl groups attached to atoms N3 and N4 are cis-configuration
to each other. The Cu–Zn separation is 2.9733(9) Å.
In order to monitor the conformational variations of DNA in
solution, CD spectroscopy of the complex in the absence and pres-
ence of calf thymus DNA (CT-DNA) was measured. The buffer used
was 5 mM Tris, 50 mM NaCl at pH 7.2 (stored at 4 °C and used
when not more than 4 days) and the CT-DNA was dissolved in it.
The spectrum of CT-DNA shows two conservative CD bands in
the UV region, a positive band at 278 nm due to base stacking
* Corresponding author. Address: Key Laboratory for Green Chemical Process of
Ministry of Education, Wuhan Institute of Technology, Wuhan 430073, PR China.
Fax: +86 27 8719 4465.
E-mail address: zhiqpan@163.com (Z. Pan).
1387-7003/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2009.12.013

Y. He et al. / Inorganic Chemistry Communications 13 (2010) 314–318
315
Scheme 1. Synthesis of ligand L and its complex.
Fig. 2. CD spectra of DNA in the absence and presence of the complex,
[DNA] = 20 M. (a) [1] = 0; (b) [1] = 100 M.
l
l
Fig. 1. Perspective view of cation of complex 1 with atomic labeling.
spectra of DNA. The concentration of CT-DNA was determined from
and a negative band at 246 nm due to polynucleotide helicity [17].
The CT-DNA spectra in the presence of the complex exhibited a sig-
nificant decrease in molar ellipticity for the positive CD band at
278 nm that indicates a distortion of the initial B-form conforma-
tion of DNA (Fig. 2), illustrating that the complex have good activ-
ity when binding with DNA and intercalation into CT-DNA [18,19]
at any rate.
its absorption intensity at 260 nm with
a molar extinction
coefficient of 6550 Mꢁ1 cmꢁ1 [20]. The absorption spectra of the
complex in the absence and presence of CT-DNA at different con-
centrations (0–140 lM) are given in Fig. 3. Intense absorption
bands observed near 350 nm was attributed to the LMCT transition
involving the phenoxide groups and the metal ions. The observed
hypochromicity and the bathochromic shifts were 85% with red
shift of 2 nm at a ratio [complex]/[DNA] of 2.5. The spectral charac-
The binding ability of the complex with calf thymus (CT-DNA) is
characterized by measuring its effects on the UV and fluorescence

316
Y. He et al. / Inorganic Chemistry Communications 13 (2010) 314–318
Fig. 3. Aborption spectra of the complex in Tris–HCl buffer upon addition of calf
thymus DNA. Lane 1: [1] = 100 M, lane 2: [1] = 100 M + [DNA] = 75 M, lane 3:
M, lane 4: [1] = 100 M + [DNA] = 60 M, lane 5,
M, lane 6: [1] = 100 M + [DNA] = 140 M.
l
l
l
Fig. 5. Emission spectra of EB bound to DNA in the absence (line 1) and presence
(solid line) of the complex [1]. [EB] = 4 M, [DNA] = 28 M, [1] = 0–30 M, respec-
tively. k = 360 nm. lines 2–4 shows the intensity changes upon increasing the
[1] = 100
[1] = 100
l
M + [DNA] = 95
l
l
l
l
l
l
l
l
lM + [DNA] = 110
l
complex concentration.
teristics suggest that the complex interacts with CT-DNA most
likely through the intercalation mode.
In order to quantitatively investigate the binding strength of the
complex with CT-DNA, the intrinsic binding constant Kb was ob-
tained by monitoring the changes in absorbance at 350 nm for
the complex as increasing concentration of CT-DNA using the
equation [21], [DNA]/Eap = [DNA]/E + 1/(KbE), where Eap
=
ea
ꢁ
ef,
E = eb f and b correspond to Aobs/[L], the extinction coeffi-
ꢁ
ef;
ea
,
e
e
cients for the free complex and the complex in the fully bound
form, respectively, and through the ratio of slope to the intercept
from the plots of [DNA]/Eap vs [DNA] (Fig. 4). The calculated Kb va-
lue was 5.0 ꢂ 105 Mꢁ1. The Kb value and hypochromism illustrate
that the complex at any rate partial intercalates into CT-DNA [22].
Fig. 5 shows the changes of the emission spectra of Ethidium
bromide (EB) bound to DNA in the absence and presence of the
complex upon increasing the complex concentrations. The results
suggest that the emission band at 605 nm of the EB–DNA system
decreased in intensity with increasing the complex concentrations,
indicating that the complex could compete with EB in binding to
DNA. According to the classical Stern–Volmer equation [23], the
plot of I0/I vs [complex] (I0 is the emission intensity of EB–DNA
in the absence of complex; I is the emission intensity of EB–DNA
in the presence of complex) is depicted in Fig. 6, and the binding
Fig. 6. Plot of I0/I vs [complex] for fluorescence titration of CT-DNA with complex.
constant 1.02 ꢂ 105 Mꢁ1 (Kb) is obtained by the ratio of the slope
to intercept. The result suggests that the interaction of the complex
with DNA is an unclassical intercalative mode. The average Kb va-
lue obtained from the above two methods are similar and the aver-
age is 3.0 ꢂ 105, which almost equals to that of typical classical
intercalators EB–DNA and much larger than those of the homodi-
nuclear complexes [24] and the homodinuclear macrocyclic com-
plexes with soft arms [9], due to the rigid furan macrocycle
ligand, which facilitate DNA binding.
To further understand the interaction mode between the com-
plex and CT-DNA, The changes in relative viscosity of CT-DNA in
the presence of the complex was measured (Fig. 7). The viscosity
of DNA decreases with increasing complex concentration, suggest-
ing that the binding mode of the complex was partially interca-
lated between DNA base-pairs and belong to
intercalative mode [25].
a unclassical
In order to evaluate the function of the complex to incise
pBR322 DNA, the cleavage reaction of supercoiled pBR322 DNA
was monitored by agarose gel electrophoresis. All experiments
were performed in TAE (40 mM Tris base, 40 mM acetic acid,
1 mM EDTA) buffer solutions (pH 7.5). Fig. 8 shows that the com-
Fig. 4. Plot of [DNA]/Eap vs [DNA] for absorption titration of CT-DNA with complex
1.

Y. He et al. / Inorganic Chemistry Communications 13 (2010) 314–318
317
Fig. 10. Anaerobic and aerobic pBR322 DNA cleavage at room temperature for 3 h
incubation. Lanes 1–3 aerobic conditions. Lanes 4–6: anaerobic conditions. Lanes
1and 4: DNA + ddH2O; lanes 2 and 5: DNA + DMF and lanes 3 and 6: DNA + 1
(100 lM).
plex through adding hydroxyl radical scavengers (5 mM DMSO)
and singlet oxygen scavenger (5 mM NaN3) in the system [20].
As shown in Fig. 9, the cleavage efficiency is not found in the pres-
ence of the scavengers, which suggest that the cleavage reaction
mediated by the complex may be caused by singlet oxygen or hy-
droxyl radical in the solution. Furthermore, the DNA cleavage
experiments of the complex in argon were carried out. Under
anaerobic conditions, no DNA degradation was observed over a
period of 3 h at room temperature, which can be seen in Fig. 10.
The result of no DNA cleavage activity in the absence of oxygen
demonstrates that oxidative cleavage occurred in this system.
In summary, we prepared and structurally characterized a new
unsymmetrical bis-furan pendant-armed macrocyclic heterodinu-
clear Cu(II)Zn(II) complex. The complex is partially intercalated
into DNA base-pairs with an unclassical intercalative mode and
exhibits efficient cleavage of supercoiled DNA (pBR 322DNA) in
the absence of any external agents via oxidative mechanism. The
DNA cleavage activity of the complex is proportional to incubation
time. The bind constant of the complex with DNA is much larger
than those of homodinuclear macrocyclic complexes.
Fig. 7. Effect of the complex on the relative viscosity of CT-DNA. [DNA] = 140 lM.
Acknowledgements
Fig. 8. Gel electrophoresis diagram showing the cleavage of pBR322 DNA at
different incubation time and the complex concentration at room temperature.
This work was supported by National Nature Science Founda-
tion of China (No. 20871097), the foundation for midlife and youth
Lanes 1–5, DNA + 1 for 5 h, 4 h, 3 h, 2 h, 1 h, respectively. (a) 50
100 M complex and (c) 200 M complex.
lM complex; (b)
l
l
excellent innovation group of Hubei Province, China (No.
T
200802), the Key Foundation of the Education Department of Hu-
bei Province, China (No. D20081503).
plex can transform the supercoiled (SC) to nicked circular (NC)
DNA forms, and the cleavage activity of the complex increased as
the enhancement of the incubation time, but was not obvious
concentration-dependent effect. Comparing the results with those
of [Cu(OP)2]2+ (OP = 1,10-phen-anthroline), the cleavage activity of
the complex was weaker than that of it [S1], which was one of the
best-characterized nucleolytic agents [26]. The known homonu-
clear complexes have DNA cleavage activities only in the presence
of H2O2 [9–11], however, the title complex has the activity without
H2O2, illustrating that the rigid furan groups in the macrocycle
ligand may have synergistic effect in the DNA cleavage.
Appendix A. Supplementary material
CCDC 665425 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via http://www.ccdc.ca-
m.ac.uk/data_request/cif. Gel electrophoresis diagram showing
the cleavage of pBR322 DNA with 1 (100
(100 M) for 3 h incubation. Supplementary data associated with
l
M) and [Cu(OP)2]2+
l
this article can be found, in the online version, at doi:10.1016/
j.inoche.2009.12.013.
Although the cleavage reaction promoted by the complex does
not require additional external agents, we carefully investigated
the effects of scavengers on the DNA cleavage ability of the com-
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